The present invention, in some embodiments thereof, relates to bispecific antibodies, monospecific, asymmetric antibodies and methods of generating same.
Bispecific antibodies (BsAbs) are antibodies with two binding sites, each directed against a different target antigen, to which they can bind simultaneously (Baeuerle and Reinhardt, 2009). This property enables the development of therapeutic strategies that are not possible with conventional monoclonal antibodies. The primary applications of bispecific antibodies include a) simultaneous inhibition of two targets (e.g. receptors of soluble ligands, a receptor and a ligand or two different ligands), b) retargeting, where one binding specificity is directed against a target cell (usually a tumor cell) whereas the other binding site is used to recruit a toxic activity or moiety to the target cell (T or NK cells; enzyme for prodrug activation; cytokine, radionuclide, virus, toxin), c) increased specificity, when strong binding mediated by simultaneous engagement of both antibody arms can only occur on cells expressing both antigens (Fischer and Leger, 2007; Amann et al., 2009; Lutterbuese et al., 2010). Since bispecific antibodies are regarded as promising therapeutic agents, several bispecific modalities have been developed, but their utility is limited due to problems with stability and manufacturing complexity. Several strategies for the creation of bispecific antibodies have been proposed over the past 20 years but despite numerous attempts and various proposed antibody formats, the BsAbs suffer from lack of product homogeneity and challenging production problems (Fischer and Leger, 2007; Chames and Baty, 2009).
Initially, attempts were made to produce bispecific antibodies by fusing two hybridomas, each producing a different antibody, resulting in what was referred to as “quadromas” or hybrid hybridomas. However, quadromas suffered from genetic instability and yielded heterogeneous mixes of the heavy and light chains. It was found that on average an at random association of L chains with H chains was found of the two antibodies, and only a tiny fraction were the desired bispecific antibodies (De Lau et al., 1991; Massino et al., 1997). If one considers creating a bispecific antibody from two monospecific antibodies, A and B, efficient assembly of a bispecific antibody in an IgG format has two basic requirements, one is that each heavy chain associates with the heavy chain of the second antibody (heavy chain A associates with heavy chain B) and no homoassociation (A+A or B+B) occurs. The second requirement is that each light chain associates with its cognate heavy chain (light chain A with heavy chain A, and not light chain B with heavy chain A or light chain A with heavy chain B). The random association of antibody chains in quadromas could not meet those requirements.
Efficient generation of bispecific antibodies was made possible by advances in antibody engineering. Advanced antibody engineering enabled the creation of new recombinant antibody formats like tandem single-chain variable fragment (scFv) (Robinson et al., 2008), diabodies (Hudson and Kortt, 1999), tandem diabodies (Kipriyanov, 2009), two-in-one antibody (Bostrom et al., 2009), and dual variable domain antibodies (Wu et al., 2007). These new antibody formats solved some of the manufacturing issues, providing homogeneous preparations. However, most of these scaffolds, due to their small size, suffer from poor pharmacokinetics and therefore require frequent dosing or conjugation to larger carrier molecules to improve half-life (Constantinou et al., 2009).
Ridgway et al., 1996 provided a solution to one of the two criteria for making bispecific antibodies making it possible to re-consider IgG-based bispecific antibodies technically feasible. They described an engineering approach termed “knobs into holes” which allows only heterodimerization between the heavy chains of “antibodies A and B” to form, disallowing homodimerization. While studying the rules for heavy chain association, the authors postulated that it is primarily dependent on interfacial interactions between the CH3 domains of the two heavy chains. When protein domains or subdomain interact, a knob is a bulky side chains that protrudes into the opposite domain where it is aligned with a small side chain that makes such invasion possible. In their approach, knob and hole variants were anticipated to heterodimerize by virtue of the knob inserting into an appropriately designed hole on the partner CH3 domain. Knobs were constructed by replacing small side chains with the largest side chains, tyrosine or tryptophan. Holes of identical or similar size to the knobs were created by replacing large side chains with the smaller ones, in this case alanine or threonine. This way, two heavy chains that are knob variants can not homoassociate because of side chain clashes, and the homoassociation of two hole variants is less favored because of the absence of a stabilizing side-chain interaction. Subsequently, this group engineered a disulfide bond near the c-terminus of the CH3 domain to further stabilize the assembled bispecific antibodies (Merchant et al., 1998).
U.S. Pat. No. 7,183,076 teaches a method of generating bifunctional antibodies using the knob and hole approach.
However, the knobs into holes approach provided a solution only for the heteroassociation of the heavy chain and did not provide one for the correct pairing of each heavy chain with its cognate light chain. Therefore, in that study, a bispecific IgG capable of simultaneously binding to the human receptors HER3 and cMpI was prepared by coexpressing a common light chain and the corresponding remodeled heavy chains followed by protein A chromatography. The engineered heavy chains retain their ability to support antibody-dependent cell-mediated cytotoxicity as demonstrated with an anti-HER2 antibody (Merchant et al., 1998).
International application 2010/115589 teaches trivalent bispecific antibodies in which to a monospecific IgG carrying knobs into holes mutations, a VH and VL of a second specificity are fused at the C-terminus of the two CH3 domains.
Similar molecules are described in U.S. Patent Application Publication No. 2010/0256340.
Disulfide-stabilized Fvs were first described by the group of Andreas Plückthun (Glockshuber et al., 1990) and later by the group of Ira Pastan (Brinkmann et al., 1993; Reiter et al., 1994a; Reiter et al., 1994b; Reiter et al., 1995). The Pastan group did extensive work on dsFvs, and used molecular modeling to identify positions in conserved framework regions of antibody Fv fragments (Fvs) that are distant from CDRs, and potentially can be used to make recombinant Fv fragments in which the unstable VH and VL heterodimer is stabilized by an engineered interchain disulfide bond inserted between structurally conserved framework positions. A disulfide bond was introduced at one of these positions, VH44-VL105 or VH111-VL48 was shown to stabilize various Fvs that retain full binding and specificity.
U.S. Pat. Nos. 5,747,654, 6,147,203 and 6,558,672 teach disulfide-stabilized Fvs, wherein the Fvs are engineered to introduce additional disulfide bonds between the light and heavy chains.
Additional background art includes Jackman et al., Journal of Biological Chemistry Vol 285, No. 27, pp. 20850-20859, Jul. 2, 2010 and Schaefer et al., Proc Natl Acad Sci USA. 2011 Jul. 5; 108(27): 11187-11192.